PACE4, a proprotein convertase (PC) studied in our laboratory has a significant role in the processing of metalloproteases and other cancer-related substrates. We have found that PACE4 is overexpressed in high grade murine skin squamous cell carcinomas induced by chemical carcinogenesis protocols. In addition, when overexpressed in mouse skin-derived keratinocytes it is able to increase cell invasiveness. In the present proposal, using transgenic mouse models and chemically-induced skin tumors, we will attempt to prove in vivo, the hypothesis that PCs contribute to tumor development and progression and that in vivo PC inhibition will result in the decrease or absence of skin tumor formation and/or aggressive malignant phenotypes. In addition, we will attempt to demonstrate which of the PC substrates, once activated in vivo, will contribute significantly to the process of tumor growth and progression. The following three aims are to be addressed: 1) Using a transgenic mouse model in which PACE4 is under the control of the keratin 5 promoter (K5-PACE4 mice), chemical carcinogenesis protocols will be used to determine the effect of PACE4 tissue specific overexpression on different stages of skin carcinogenesis. We expect that these transgenic animals will exhibit an increased susceptibility to carcinogenesis. 2) Using the transgenic models, inhibitors of PCs will be evaluated in vivo. We will establish the feasibility of a genetic transfer approach by generating transgenic mice expressing the protein-based PC inhibitor alpha-1 PDX (PDX) in the epidermis. These mice should be resistant to chemical carcinogenesis and/or tumor progression and should neutralize the effects of PACE4. In addition, we will use known PC inhibitors such as chloromethyl ketone, applied topically to the skin of K5-PACE4 mice to evaluate the reversal of the PACE4-induced susceptibility to skin chemical carcinogenesis. 3) In order to determine the critical in vivo PC substrates for cancer development, we propose to cross K5-PACE4 mice with other transgenic animals expressing PACE4 substrates targeted to the skin such as IGF-1, TGF-beta and MT2-MMP. This approach will establish the in vivo hierarchy of the respective substrate activation in the acceleration of skin tumor development.